Novel prenyl transferase inhibitors

ABSTRACT

Farnesyl diphosphate analogs, specifically the 3-substituted alcohol precursors of the diphosphate analogs, 3-allylfarnesol and 3-vinylfarnesol, are potent inhibitors of mammalian protein fanesyltransferase (FTase). 3-allylgeranylgeraniol is a highly specific cellular inhibitor of protein geranylgeranylation (GGTase I). Furthermore, these compounds are able to efficiently block the anchorage-dependent growth of ras transformed cells. While 3-allylfarnesol inhibits protein farnesylation in situ, 3-vinylfarnesol instead leads to the abnormal prenylation of proteins with the 3-vinylfarnesyl group. In a similar manner, treatment with 3-allylgeranylgeraniol inhibits protein geranylgeranylation while 3-vinylgeranylgeraniol restores protein geranylgeranylation in cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/089,625 filed on Jun. 16, 1998. This application is acontinuation of co-pending U.S. Ser. No. 09/334,704 filed on Jun. 17,1999.

STATEMENT GOVERNMENT RIGHTS

[0002] This invention has been made with Government support undercontract no. CA-67292 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates generally to novel analogs of farnesol andgeranylgeraniol, and more specifically to 3-substituted farnesol andgeranylgeraniol analogs, that block the prenylation of proteins incells.

[0005] 2. Background of the Prior Art

[0006] Proteins are modified by a mevalonate pathway intermediate. Thereare three different protein prenylation motifs in thispathway—farnesylation, geranylgeranylation, and bis-geranylgeranylation.The first modification is carried out by an enzyme, protein farnesyltransferase (FTase), which recognizes the CAAX box (where A=aliphaticamino acid and X=Ser or Met) at the carboxyl terminus of the proteinsubstrate and then attaches the farnesyl group from farnesyl diphosphate(FPP) to the free sulfhydryl of the cysteine residue. FIG. 1 is areaction pathway for protein farnesylation of, illustratively, a Rasprotein. The second, closely related enzyme, proteingeranylgeranyltransferase I (GGTase I), attaches a geranylgeranyl moietyfrom geranylgeranyl diphosphate (GGPP) to a cysteine in a similar CAAXbox, where leucine (X=Leu) is the carboxyl terminal residue. The thirdenzyme, GGTase II, attaches two geranylgeranyl residues to two cysteineresidues at the carboxyl terminus of Rab proteins.

[0007] Once initial studies demonstrated that the key signaltransduction protein and oncogene product Ras is farnesylated, FTasebecame the subject of intense research interest on the basis thatinhibitors of this enzyme could block the action of mutant Ras proteinsand halt the growth of ras-transformed cells, and therefore would bepotential anti-cancer agents. Mutant forms of Ras proteins are involvedin about 30% of human cancers. These include pancreatic adenocarcinomas,colon adenocarcinomas, and adenomas; thyroid carcinomas and adenomas;lung adenocarcinomas; myeloid leukemia; and melanomas. Therefore, thereis a need in the art for an FTase inhibitor that can block the growth ofras-transformed cells in vivo.

[0008] Significant progress has been made in the development ofpeptide-based FTase inhibitors, and some of these compounds have showngreat promise in vivo as potential anti-cancer agents. Merck hasreported a peptidomimetic inhibitor that is effective in vivo in a mousepancreatic carcinoma model. However, the peptide-based inhibitors arecomplicated molecules that require numerous synthetic steps to prepare.This limits their availability and increases cost. Moreover, thepeptidomimetic inhibitors are not able to penetrate cell membranes dueto their polarity. Therefore, in order to be administered in vivo, thesedrugs must be converted to a prodrug ester form. Even in the prodrugform, the in situ (cell culture) and in vivo potency is several ordersof magnitude less than the potency of the parent inhibitor and theisolated enzyme. Furthermore, since peptide-based drugs are usuallypolar, they are susceptible to proteolytic degradation. This preventsthe compound, even in its prodrug form, from being given orally, therebynecessitating dosing by i.v. administration.

[0009] While the specificity of FTase for its protein substrate has beenextensively explored, there have only been limited reports on itsspecificity for farnesyl pyrophosphate or farnesyl diphosphate (FPP;Compound 10 on FIG. 1). FPP is a biosynthetic intermediate that occupiesa key branch point in the mevalonate pathway. The primary route for FPPmetabolism is its conversion into squalene by the enzyme squalenesynthase. Squalene is then transformed by a series of enzymatic steps tocholesterol. Squalene synthase has attracted significant interest as apotential additional target for cholesterol-lowering agents. FPP is alsoconverted in the cell to other important isoprenoids, such as dolicholand ubiquinone, which are utilized in protein glycosylation and electrontransport, respectively. Most recently it has been recognized that FPPplays an additional crucial role in the cell. It is utilized by theenzyme protein farnesyl transferase (FTase) as the source of a farnesylmoiety that is attached to the cysteine sulfhydryl on the ras G proteinsand certain other proteins which bear a carboxyl-terminal Cys-AAX—OHsequence. This farnesylation event and the subsequent proteolysis andcarboxymethylation modifications serve to increase the hydrophobicity ofthese proteins, and thus, convert them to peripheral membrane proteins.There is, therefore, a need for FPP-based FTase inhibitors as probes foranalyzing the FPP-binding site of FTase.

[0010] Novel FPP analogs were synthesized as probes of the FPP-bindingsite of FTase and characterized by their interaction with recombinantyeast FTase (yFTase). The vinyl analog, 3-vinyl farnesyl diphosphate(3-vFPP; FIG. 1, Compound 10a) was designed as a potentialmechanism-based inhibitor, but instead was a poor alternative substratefor yFTase. These results were reported in Gibbs, et al., J. Org. Chem.,Vol. 60, pages 7821-7829 (1995). In contrast, a sterically encumberedanalog, 3-tert-butyl farnesyl diphosphate (3-tbFPP; FIG. 1, Compound10b), is an exceptionally poor substrate and a potent competitiveinhibitor of this enzyme. See, Mu, et al. J. Org. Chem., Vol. 61, pages8010-8015 (1996).

[0011] The results obtained by the various prenyl transferase inhibitorsproposed in the art demonstrate unpredictability with respect toinhibition of FTase or geranylgeranylase (GGTase) in vitro and in vivo.There is, thus, a need for prenyl transferase inhibitors that areeffective against mammalian FTase, which is the clinically relevantvariant of the enzyme, both in vitro for research purposes and in vivofor therapeutic purposes.

[0012] It is, therefore, an object of this invention to provide novelprenyl transferase inhibitors that are easier to prepare than otherfarnesyl analog FTase inhibitors.

[0013] It is also an object of this invention to provide novel prenyltransferase inhibitors that exhibit better cellular penetration.

[0014] It is another object of this invention to provide novel prenyltransferase inhibitors that are useful as anticancer agents.

[0015] It is a still further object of this invention to provide novelprenyl transferase inhibitors that are bioavailable and non-toxic to thehost and that are cytotoxic as compared to other FTase inhibitors thatare merely cytostatic.

[0016] It is yet another object of this invention to provide novelgeranylgeranyl transferase inhibitors that are potentially useful asrestenosis inhibitors.

SUMMARY OF THE INVENTION

[0017] The foregoing and other objects are achieved by this inventionwhich comprises, in a composition of matter embodiment, 3-substitutedisoprenol analogs that block the prenylation of proteins in cells. Inpreferred embodiments, the isoprenol analogs are analogs of thealcohols, farnesol and geranylgeraniol, as well as their diphosphatederivatives. The active, diphosphate derivatives of these compounds arepotent inhibitors of mammalian protein prenyltransferases.

[0018] The alcohol precursors efficiently block the growth ofanchorage-independent ras-transformed cells.

[0019] The farnesol and geranylgeraniol analogs within the contemplationof this invention, include without limitation,

[0020] Formula (I):

[0021] wherein R′ is a C₁₀-C₂₀ saturated or unsaturated alkyl, aryl,heteroaryl, or cycloalkyl which, in some embodiments, includesubstituents, some of which may contain heteroatoms, such as N, O, S,and F; R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group which may be substituted; andX is —OH or —P₂O₇. As used herein, the composition includes enantiomers,stereoisomers, and geometric isomers.

[0022] In preferred embodiments, R′ is geranyl and farnesyl and R isselected from the group vinyl, ethyl, allyl, saturated and unsaturatedisomers of propyl, butyl, and pentyl, cyclopropyl, isobutyl,cyclopentyl, phenyl and heterosubstituted moieties, such asfluorophenyl, (trimethylsilyl)methyl, 1-ethoxyvinyl, and 2-furanyl,2-thiophenyl.

[0023] In a preferred embodiment, the 3-substituted farnesol orgeranylgeraniol analogs have of the following formulae (II) and (III):

[0024] Formula(II):

[0025] Formula (III):

[0026] wherein R is preferably vinyl, allyl, isopropyl, cyclopropyl,isobutyl, cyclopentyl, or phenyl. However, R may comprises othersubstituted or unsubstituted C₂-C₁₀ saturated and unsaturated alkyl,aryl, and cycloalkyl groups, C₆-C₁₀ aromatic groups, and heteroaromaticgroups, some of which are illustratively shown in FIG. 2B. As statedabove, the isoprenyl alcohols are prodrugs for the active diphosphateform where R is methyl.

[0027] Illustrative embodiments of the 3-substituted farnesol andgeranylgeraniol analogs are shown on FIGS. 2A and 2B. The compounds areidentified on FIGS. 2A and 2B by their common name. Those compounds thatare specifically referenced in this application are identified bycompound number, and if abbreviated herein, the abbreviation.

[0028] In particularly preferred embodiments, the 3-substitutedcompounds are 3-vinyl farnesol, 3-allylfarnesol, 3-isopropylfarnesol,3-vinyl geranylgeraniol, 3-allylgeranylgeraniol, and3-isopropylgeranylgeraniol. In the studies reported herein,3-allylfarnesol inhibits protein farnesylation in situ and3-vinylfarnesol instead leads to the abnormal prenylation of proteinswith the 3-vinylfarnesyl group. In a similar manner, treatment with3-allylgeranylgeraniol inhibits protein geranylgeranylation while3-vinylgeranylgeraniol restores protein geranylgeranylation in cells.Furthermore, 3-allylgeranylgeraniol is one of the most potent inhibitorsof GGTase I discovered to date. Studies have now confirmed that the3-allyl and 3-vinyl analogs are potently cytostatic and cytotoxic tohuman pancreatic cancer cells (HPAC) in situ. In addition, the 3-allylanalogs have been shown to exhibit selective cytostatic effects on amouse colon 38 tumor cell line at levels where the same amount has noeffect on human fibroblasts. Moreover, preliminary animal studies withsevere combined immunodeficiency (SCID) mice indicate that thesecompounds are non-toxic.

[0029] As indicated above, the isoprenol derivatives, 3-substitutedfarnesol or geranylgeraniol, are precursors, or prodrugs, and as such,rely on the target cells to carry out the biological activation.Therefore, the isoprenol derivative are administered as an anticanceragent, specifically as a prodrug for the active diphosphate, to inhibitthe respective prenyl transferases in cells. In particular, inhibitionof FTase reduces the level of protein farnesylation in a host, and hencereduces the activity of proteins, such as Ras proteins, which requirefarnesylation for activation. Of course, these compounds can exert amultitude of other beneficial effects, both known and yet to bediscovered.

[0030] In a method of use embodiment, a method of treating cancer,specifically cancers of the type which are susceptible to treatment byprenyl transferase inhibitors, comprises administering an effectiveamount of at least one 3-substituted isoprenol derivative. Inparticularly preferred embodiments, the 3-substituted isoprenolderivative is selected from the group consisting of 3-allyl farnesol,3-vinyl farnesol, 3-allyl geranylgeraniol, and 3-vinyl geranylgeraniol.These cancers include any cancer having tumors which are associated withabnormal activity of oncogenes in the ras family, including the threemammalian ras genes, H-ras, K-ras, and N-ras. Other ras proteins includethose whose DNA coding regions hybridize to the coding regions of knownras genes. Abnormal ras activity is associated with 30-50% of all lungand colorectal carcinomas and up to 95% of pancreatic carcinomas. Ofcourse, the type of cancers susceptible to prenyl transferase inhibitorsis not limited to those bearing ras mutations. Protein prenylation isrequired for the activity of several signal transduction pathways. Thus,the 3-substituted isoprenol derivatives of the present invention may beused to treat cancers bearing mutations in other oncogenes, includingbut not limited to various growth factor receptor genes, rho genes, andPTPCAAX genes. Other farnesyl transferase inhibitors have proven to beeffective at blocking the growth of cancer cells that do not bear a rasmutation, as well as those that do. See, Cox, et al., Biochim. Biophys,Acta, Vol. 1333, pages F51-F71 (1997).

[0031] The novel analogs may be administered alone or in conjunctionwith other drugs. The analogs may be administered in a variety of ways,orally, parenterally, topically, etc. In injectable forms, the analogsmay be delivered subcutaneously, intraperitonealy, or intravascularly.Of course, the analogs may be combined with a delivery vehicle and otherfillers, excipients, and the like, as are known in the art to form apharmaceutical dosage form. Illustrative dosage forms include, tablets,capsules, gels, suspensions, emulsions, liposomes, nanoparticles, etc.Oral dosage forms may be encapsulated or coated, as necessary, in anymanner which is known in the art, to protect and/or release the activeagent at the appropriate point in the gastrointestinal system.

[0032] It has been reported that inhibition of proteingeranylgeranylation causes superinduction of nitric oxide synthase(NOS-2) by interleukin-1β in vascular smooth muscle cells andhepatocytes. Finder, et al., J. Biol. Chem., Vol. 272, No. 21, pages13484-13488 (1997). This indicates that the 3-substitutedgeranylgeraniol analogs may inhibit restenosis following angioplasty orother surgical intervention, such as catheterization. It is therefore,contemplated within the invention, that the 3-substitutedgeranylgeraniol derivatives may be administered to a mammal to inhibitrestenosis.

[0033] It has also been reported that farnesol inhibits Ca⁺² signals inarteries and vascular smooth muscle cells and possesses Ca⁺² channelblocker properties. Roulle, et al., J. Biol. Chem., Vol. 272, pages32240-32246, Dec. 19, 1997. Therefore, it is also contemplated that thenovel 3-substituted isoprenol derivatives disclosed herein may beadministered to a mammal for their vasoactive properties.

BRIEF DESCRIPTION OF THE DRAWING

[0034] Comprehension of the invention is facilitated by reading thefollowing detailed description, in conjunction with the annexed drawing,in which:

[0035]FIG. 1 is a reaction pathway for protein farnesylation of a Rasprotein;

[0036]FIGS. 2A and 2B illustrate chemical formulae of illustrative3-substituted farnesol and geranylgeraniol analogs in accordance withthe present invention;

[0037]FIG. 3A is a synthetic pathway for producing a 3-substitutedisoprenyl diphosphate, illustratively 3-vinylfarnesyl diphosphate(3-vFPP);

[0038]FIG. 3B is a synthetic pathway for producing a 3-allylfarnesyldiphosphate;

[0039]FIG. 4 is a cuprate-mediated synthetic pathway for producing3-tert-butylfarnesyl diphosphate;

[0040]FIG. 5 is a reaction route for synthesizing 3-vinyl geranylgeranyldiphosphate (3-vGPP) and the corresponding 3-vinyl geranylgeraniol;

[0041]FIG. 6 is a Western blot showing the subcellular labeling of H-Rasin H-Ras-F cells;

[0042]FIG. 7 is a Western blot showing the subcellular labeling of H-Rasof the H-Ras-GG cells;

[0043]FIG. 8 is shows the incorporation of a radiolabel into H-ras-Fcells;

[0044]FIG. 9 shows photographs of the morphology of H-Ras-F cellstreated with lovastatin and the indicated FPP analogs; and

[0045]FIG. 10 shows photographs of the morphology of H-Ras-GG cellstreated with lovastatin and the indicated GGPP.

DETAILED DESCRIPTION

[0046] Illustrative methods of making the 3-substituted isoprenoidanalogs of the present invention are set forth hereinbelow:

[0047] Stereoselective Synthesis of Farnesyl and Geranylgeranyl Analogs:

[0048] A stereoselective route to the 3-substituted farnesyl orgeranylgeranyl analogs is illustrated by the production of13-methylidenefarnesyl diphosphate, or 3-vinylfarnesyl diphosphate(3-vFPP; Compound 19) which is the active form of the desired alcoholprecursor 3-vinyl farnesol (Compound 17). The synthetic sequence, shownin FIG. 3A, includes the stereoselective Pd(0)/Cu(I)-catalyzed couplingof a vinyl triflate with an organostannane reagent, vinyltributyltin, toobtain the desired Z isomer of the divinyl ester. The divinyl ester isreduced to the corresponding alcohol. The alcohol is diphosphorylated,illustratively through a synthetic route from the alcohol into anallylic chloride which is treated with tris(tetrabutylammonium)hydrogendiphosphate to yield the desired diphosphate.

[0049] By using a polar aprotic solvent, illustratively dimethylformamide (DMF), instead of THF, in the synthesis of the triflate(Compound 15) from the β-ketoester (Compound 14), the opposite geometricisomer of the triflate, and consequently the alcohol and thediphosphate, is obtained.

[0050] The general methods disclosed herein can be adapted by those ofskill in the art to produce a multitude of compounds, including thestereoselective production of a broad range of substituted allylicalcohols. By selecting the appropriate starting compounds, farnesols andgeranylgeraniols substituted at positions other than 3-, can besynthesized. Illustrative examples, are 7-allylfarnesol andpara-biphenylfarnesol shown on FIG. 2B.

[0051] The 3-ethyl and 3-phenyl farnesol analogs were produced by thesame procedure using the appropriate organostannane. The 3-tert-butyland 3-cyclopropyl farnesyl and geranylgeranyl analogs were synthesizedusing a cuprate coupling procedure, which is illustrated herein by themethod of producing 3-tert-butyl farnesol.

[0052] As illustrated below, by the methods of making 3-vinylgeranylgeraniol and 3-allyl geranylgeraniol, the geranylgeranyl analogsmay be produced in a similar manner starting with the appropriatetriflate.

[0053] I. 3-Substituted Farnesyl Analogs

[0054] a. Vinyl Analogs:

[0055] Referring to FIG. 3A, the detailed procedures for making 3-vinylfarnesol and 3-vinyl farnesyl diphosphate (3-vFPP) are set forth below:

Ethyl 7,11-dimethyl-3-oxododeca-6,10-dienoate (Compound 14)

[0056] The sodium salt of ethyl acetoacetate (Compound 11; 20.0 mmol,3.04 g) was dissolved in 40 ml of tetra hydrofuran (THF) (distilled fromsodium/benzophenone ketyl) and cooled to 0° C. n-Butyllithium (2.0 M incyclohexane, 21.0 mmol, 10.6 ml) was added dropwise to the cooledsolution to form a solution of the dianion Compound 12. After 20minutes, geranyl bromide (Compound 13; 10.0 mmol, 1.98 ml, 2.16 g) wasadded to the solution of dianion and stirring was continued for anadditional 30 minutes at 0° C. The reaction mixture was poured into acold saturated solution of potassium hydrogen phosphate and extractedwith ether (3×20 ml). The combined organic layers were washed with water(20 ml), dried over MgSO₄, filtered, and concentrated. Flashchromatography (9:1 hexane/ethyl acetate) afforded 2.05 g (77% yield) ofCompound 14 as an oil.

Ethyl3-(((trifluoromethyl)sulfonyl)oxy)-7,11-dimethyldodeca-2(Z),6(E),10-trienoate(Compound 15)

[0057] A solution of the β-keto ester Compound 14 (4.0 mmol, 1.064 g) inTHF (10 ml; distilled from sodium/benzophenone ketyl) was added topotassium bis(trimethylsilyl)amide (0.5 M in toluene, 4.8 mmol, 9.6 ml)at −78° C. N,N,-bis(Trifluoromethanesulfonyl-N-phenylamine (4.8 mmol,1.72 g) was added and the mixture was allowed to warm to roomtemperature overnight. The mixture was taken up in 30 ml of ether andwashed with a 10% citric acid solution (2×20 ml) and water (1×20 ml).The ether layer was dried over MgSO₄, and the solvent removed in vacuo.Purification by flash chromatography (95:5 hexane/ethyl acetate) gave0.845 g (53 % yield) of the vinyl triflate, Compound 15, as an oil.

Ethyl 3-vinyl-7,11-dimethyldodeca-2(Z),6(E), (Compound 16) 10-trienoateand its isomer Ethyl-3-vinyl-7,11-dimethyldodeca-2(E),6(E),10-trienoate

[0058] Triflate Compound 15 (1.09 mmol, 434 mg), triphenyl arsine(Ph₃As; 0.11 mmol, 34 mg), bis(benzonitrile)palladium (II) chloride(0.054 mmol, 21 mg), and CuI (0.11 mmol, 21 mg) were placed in anargon-flushed flask and dissolved in NMP (1.1 ml). Vinyltributyltin (1.3mmol, 412 mg, 0.38 ml) was then added, and the reaction mixture wasstirred for about 15 hrs at room temperature. The mixture was thendissolved in 1:1 EtOAc/hexanes (100 ml), washed with aqueous KF (2×30ml) and water (20 ml), dried over MgSO₄, and then filtered andconcentrated. Purification by flash chromatography (98:2 hexane/EtOAc)afforded the desired vinyl ester (234 mg; 78% yield). The ratio of Eisomer to Z (Compound 16) isomer was determined to be 94:6 byintegration of the NMR peaks at ∂ 7.74 and 6.332.

3-Vinyl-7,11-dimethyldodeca-2(Z),6(E),10-trien-1-ol (Compound 17)

[0059] A solution of the vinyl ester Compound 16 (0.85 mmol, 234 mg) intoluene (4.2 ml; HPLC grade dried over 4 Å sieves) was treated at −78°C. under argon with diisobutylaluminum hydride (DIBAH; 1.0 M in toluene;2.38 mmol, 2.38 ml). After the addition of the hydride, the mixture wasstirred for 1 hour at −78° C. The reaction was quenched by adding thereaction mixture to saturated aqueous potassium sodium tartrate (40 ml).The organic phase was separated, and the aqueous phase was extractedwith ethyl acetate (3×30 ml). The combined organic layers were washedwith water (20 ml) and brine (20 ml) and dried (MgSO₄). Filtration andconcentration followed by flash chromatography (9: 1 hexane/ethylacetate) gave 173 mg (86%) of vinyl alcohol, 3-vinyl farnesol (Compound17).

1-Chloro-3-vinyl-7,11-dimethyldodeca-2(Z),6(E),10-triene (Compound 18)

[0060] N-Chlorosuccinimide (NCS; 0.42 mmol, 60 mg) was dissolved in 1.75ml of methylene chloride (CH₂Cl₂; distilled from CaH₂). The resultingsolution was cooled to −30° C. in a dry ice/acetonitrile bath. Dimethylsulfide (DMS; 0.45 mmol, 0.03 ml, 27 mg) was added dropwise by asyringe, and the mixture was warmed to 0° C., maintained at thattemperature for 5 minutes, and then cooled to −40° C. The resultingmilky white suspension was added dropwise to a solution of the vinylalcohol Compound 17 (0.38 mmol, 90 mg) in 5 ml of distilled CH₂Cl₂. Thesuspension was warmed to 0° C. and stirred for 2 hours. The ice bath wasremoved and the reaction mixture was allowed to warm to room temperatureand stirred for an additional 15 minutes. The resulting solution waswashed with hexane (2×20 ml). The hexane layers were then washed withbrine (2×10 ml) and dried over of MgSO₄. Concentration afforded 74 mg(77% yield) of the vinyl chloride Compound 18 as an oily liquid whichwas used directly in the next step without purification.

3-Vinyl-7,11-dimethyldodeca-2(Z),6(E),10-triene 1-Diphosphate(13-Methylidenefarnesyl Diphosphate) (3-vFPP, Compound 19)

[0061] Tris (tetrabutylammonium) hydrogen pyrophosphate (0.40 mmol, 365mg) was dissolved in acetonitrile (3 ml; freshly distilled from P₂O₅)under an argon atmosphere. Vinyl chloride Compound 18 (0.10 mmol, 25 mg)was added to the resulting milky white suspension. The mixture wasstirred at room temperature for 2.5 hours, and the solvent was removedin a rotary evaporator at room temperature. The residue was dissolved indeionized water, and the resulting solution was passed through a 2×8 cmDowex AG50×8 ion exchange column (NH₄ ⁺ form). The eluant was thenconcentrated in vacuo to yield a pale yellow solid which was thendissolved in 2 ml of 25 mM ammonium bicarbonate. The resulting mixturewas purified by reverse-phase HPLC using a program of 5 minutes of 100%A followed by a linear gradient of 100% A to 100% B over 30 minuteswherein A is 25 mM aq. NH₄HCO₃ (pH 8.0) and B is CH₃CN; column,WaterskiBondapak C₁₈ 25 mm×100 mm Radial-Pak cartridge; flow rate, 5 ml;UV monitoring at 214 and 230 nm. The retention time of the diphosphateCompound 19 was 25 minutes. The fractions containing the product werepooled, the acetonitrile was removed by rotary evaporation resulting inpure Compound 19 as a white, fluffy solid (88% yield).

[0062] b. Allyl Analogs:

[0063] Referring to FIG. 3B, certain details of the procedures formaking 3-allyl farnesol and 3-allyl farnesyl diphosphate (3-alFPP) areset forth below:

Ethyl 3-allyl-7,11-dimethyldodeca-2(Z),6(E),10-trienoate (Compound 40)

[0064] Ethyl3-(((trifluoromethyl)sulfonyl)oxy)-7,11-dimethyldodeca-2(Z),6(E),10-trienoate(Compound 15; 317 mg; 0.79 mmol); triphenylarsine (25 mg; 0.82 mmol),bis(benzonitrile)palladium (II) chloride (15.3 mg; 0.39 mmol); andcopper iodide (15.3 mg; 0.085 mmol) were placed in an argon-flushedflask and dissolved in 1.0 ml N-methylpyrrolidone (NMP). Once dissolved,allyltributyltin (534 mg; 0.5 ml; 1.61 mmol) was added dropwise and thereaction mixture was stirred for about 24 hours at 100° C. The mixturewas then taken up in a 1:1 solution of hexanes/EtOAc (100 ml), washedwith a 10% KF solution (2×30 ml) and water (20 ml), and then dried overMgSO₄, filtered and concentrated. Purification by flash chromatographyusing a 98:2 hexanes/EtOAc solvent system yielded 73 mg (31% yield) ofthe desired allyl ester, ethyl3-allyl-7,11-dimethyldodeca-2(Z),6(E),10-trienoate.

3-Allyl 7,11-dimethyldodeca-2(Z),6(E),10-trien-1-ol (Compound 41)

[0065] A solution of the allyl ester, ethyl3-allyl-7,11-dimethyldodeca-2(Z),6(E),10-trienoate (100 mg; 0.343 mmol),in 2.4 toluene was cooled to −78° C. under an argon atmosphere.Diisobutylaluminum hydride (1.0 M in toluene; 1.5 ml; 9.04 mmol) wasthen added and the reaction mixture was stirred at −78° C. for one hour.The reaction was quenched by adding the solution to saturated aqueouspotassium sodium tartrate 930 ml. The organic phase was separated andthe aqueous layer was extracted with ethyl acetate (3×20 ml). Theorganic layers were combined, washed with water (10 ml) and NaCl (10 ml)and dried over MgSO₄, filtered, and concentrated. Purification by flashchromatography using a 9:1 hexanes/EtOAc solvent system gave 68 mg(75.8% yield) of the desired allyl alcohol (3-allylfarnesol; Compound41) as an oil.

1-Chloro-3-allyl-7,11-dimethyldodeca-2(Z),6(E),10-triene (Compound 42)

[0066] NCS (60 mg; 0.42 mmol) was dissolved in 1.75 ml of CH₂Cl₂ and theresulting solution was cooled to −30° C. Dimethyl sulfide (27 mg; 0.03ml; 0.45 mmol) was then added dropwise and the mixture was warmed to 0°C. and maintained at that temperature for 5 min then taken back down to−40° C. The allyl alcohol (68 mg; 0.261 mmol) dissolved in 5 ml CH₂Cl₂was added dropwise to the resulting mixture. The suspension was againwarmed to 0° C. and stirred for 2 hrs. The ice bath was removed, and thereaction mixture was allowed to warm to room temperature. At roomtemperature, the mixture was stirred for an additional 15 minutes. Themixture was washed with hexanes (2×15 ml) and the hexane layers werewashed with brine (2×10 ml), dried over MgSO₄, filtered, andconcentrated via a rotary evaporator to give 24 mg (33% yield) of thedesired allyl chloride as an oil. Purification was not necessary for usein the next step.

3-Allyl-7,11-dimethyldodeca-2(Z),6(E),10-triene 1-diphosphate (Compound43)

[0067] Tris(tetrabutylammonium)hydrogen pyrophosphate (365 mg; 0.40mmol) was dissolved in 3 ml acetonitrile under an argon atmosphere. Theallyl chloride (24 mg;0.086 mmol) was then added and the reaction wasallowed to stir at room temperature for 2.5 hrs. The solvent was thenremoved via rota-vapping and the residue was dissolved in deionizedwater and passed through a 2×8 cm Dowex AG5O×8 ion exchange column (NH₄⁺ form). The eluant was concentrated by lyophilization and wasimmediately dissolved in 2 ml of a 25 mM ammonium bicarbonate solution.This mixture was purified by reverse-phase HPLC using a program of 5 minof 100% A followed by a linear gradient of 100% A to 100% B over aperiod of 30 min.(A is 25 mM ammonium bicarbonate (pH 8.0); B is CH₃CN;column was Waters μBondapak 25 mm×100 mm Radial-Pak cartridge; flowrate, 3 ml; UV monitoring at 21 4 nm and 230 nm). The fractionscontaining the product (retention time was 17 min.) were collected andthe acetonitrile was removed by rotary evaporation at room temperature.The aqueous layer was lyophilized to give the 3-allylFPP (Compound 43;see also FIG. 1, Compound 1c; 31.5 mg; 90% yield) as a pure white solid.

[0068] C. tert-Butyl Analogs

[0069]FIG. 4 illustrates a cuprate-mediated synthesis of3-tert-butylfarnesyl diphosphate (3-tbFPP; Compound 25)

Ethyl 3-tert-butyl-7,11-dimethyldodeca-2(Z),6(E),10-trienoate (Compound22)

[0070] CuCN (0.47 mmol, 42 mg) and 1.0 ml of ether (distilled fromNa/benzophenone) were placed in a flame-dried argon-flushed flask toform a slurry. The resulting slurry was cooled to −78° C. and thentert-butyllithium (1.7 M in pentane, 0.47 mmol, 0.28 ml) was addeddropwise. The mixture was allowed to warm to 0° C. and then re-cooled to−78° C. A solution of the triflate, Compound 21 (0.32 mmol, 129 mg), in1.0 ml of ether was added dropwise and the reaction mixture was stirredfor 1 hour at −78° C. The mixture was warmed to 0° C. and quenched with2 ml of saturated NH₄Cl. The organic layer was separated and the aqueouslayer was extracted with ether (3×15 ml). The combined organic layerswere dried over MgSO₄ and concentrated in vacuo. Flash chromatography(20:1 hexanes/ethyl acetate) yielded Compound 22 as a colorless oil (67mg, 68% yield).

[0071] Compound 22 can be produced in 91% yield by substitutingtert-butylmagnesium chloride for tert-butyllithium in this procedure.This alternative method can be used successfully for the preparation ofa variety of other 3-substituted isoprenoid esters, and consequently,for the preparation of corresponding 3-substituted isoprenoid alcohols.

3-tert-Butyl-7,11-dimethyldodeca-2(Z),6(E),10-trien-1-ol (Compound 23)

[0072] Ester Compound 22 (0.14 mmol, 42 mg) was treated with DIBAH (0.1M in toluene, 0.35 mmol, 0.35 ml) at −78° C. under argon for 1 hour.Work-up and purification, as described above with respect to the 3-vinylfarnesol, afforded 33 mg (89% yield) of Compound 23, 3-tert-butylfarnesol, as a colorless oil.

[0073] In all of the synthetic routes proposed herein for producing theactive, prenyl diphosphate analogs related to the 3-substituted prenylalcohol derivatives, the alcohol is converted to the correspondingchloride and then to the corresponding diphosphate. Referring to FIG. 4,for example, the alcohol, 3-tert-butyl farnesol is converted to3-tert-butyl-2(Z),6(E),10-trien-1-chloride (Compound 24) according tothe following general procedure for the preparation of chlorides. Thechloride is converted to the diphosphate,3-tert-butyl-2(Z),6(E),10-triene diphosphate (3-tbFPP; Compound 25). Thegeneral procedure for converting the chloride to the diphosphatefollows.

[0074] General Procedure for Preparation of Chlorides

[0075] In a flame-dried, round-bottomed flask were placed withN-chlorosuccinimide (1.2 equivalents) and dichloromethane (distilledfrom CaH₂). The solution was cooled to −30° C. in an acetonitrile/dryice bath. Dimethyl sulfide (1.5 equivalents) was added dropwise to thecold solution, and the resulting milky white mixture was warmed to 0° C.for 5 minutes and re-cooled to −30° C. A solution of 1 equivalent of thealcohol in 1 ml of dichloromethane was added dropwise to the mixture at−30° C. The reaction was slowly warmed up to 0° C. and stirred for anadditional hour at that temperature. The resulting clear, colorlesssolution was stirred at room temperature for 20 minutes and poured into10 ml of cold brine solution. The aqueous layer was extracted with 2×15ml hexanes, and the combined organic layers were washed with 10 ml ofcold brine solution and dried over MgSO₄. Concentration (rotaryevaporation followed by high vacuum at room temperature) afforded thechlorides as colorless or pale yellow oils which were used directly forthe next reaction.

[0076] General Procedure for Preparation of Diphosphates

[0077] In a flame-dried, round-bottom flask were placed two equivalentsof Tris(tetra-n-butylammonium)hydrogen pyrophosphate and 1.0 ml ofacetonitrile (distilled from P₂O₅). The mixture was cooled to 0° C. and1 equivalent of chloride in 0.5 ml of acetonitrile was added dropwise.The reaction was allowed to stir at room temperature for two hours, andthe solvent was removed by rotary evaporation at room temperature. Theresidue was dissolved in 1-2 ml of ion exchange buffer (1:49 v/visopropyl alcohol and 25 mM NH₄HCO₃) and was passed through a columncontaining 3-10 ml cation-exchange resin (Dowex AG 50W-X8, NH₄ ⁺ Form).The column was eluted with two column volumes of ion exchange buffer ata flow rat of ˜1 ml/min. The eluant was dried by lyophilization and apale yellow solid was obtained. The crude product was dissolved in 1-3ml of 25 mM NH₄HCO₃ and purified by reverse-phase HPLC using a programof 5 mm of 100% A followed by a linear gradient of 100% A to 100% B over30 min. (A: 25 mM aqueous NH₄HCO₃, pH 8.0; B: CH₃CN; Vydac pH-stable C₈4.6 mm×250 mm column flow rate: 1.0 ml UV monitoring at 214 and 254 μm).The fractions were collected, pooled and dried by lyophilization, andthe diphosphates were obtained as white fluffy solids.

[0078] d. Cyclopropyl Analogs:

Ethyl 3-cyclopropyl-7,11-dimethyldodeca-2(Z),6(E),10-trienoate

[0079] To a solution of cyclopropyl bromide (0.38 mmol, 31 μl) in 1.0 mlether was added tert-butyllithium (1.7 M in pentane, 0.77 mmol, 0.45 ml)under argon at −78° C. The resulting solution was stirred for 15minutes. Triflate (Compound 21; 0.26 mmol, 102 mg) in 1 ml ether wasadded to the mixture at that temperature with stirring for 1.5 hours.Workup and purification as described above afforded 53 mg (71% yield) ofβ-keto ester as a colorless oil.

3-Cyclopropyl-7,11 -dimethyldodeca-2(Z),6(E),10-trien-1-ol

[0080] To a solution of the β-keto ester produced in the preceding step(0.22 mmol, 6 mg) in 1.0 ml of toluene (HPLC grade, stored over 4 Åsieves) was added DIBAH (1.0 M in toluene, 0.54 mmol, 0.54 ml) at −78°C. under argon. After being stirred for 1 hour at this temperature, thereaction was quenched with saturated sodium potassium tartrate (20 ml).The organic layer was separated, and the aqueous phase was extractedwith ethyl acetate ( 3×15 ml). The combined organic layers were washedwith water (2×15 ml), dried over MgSO₄, and concentrated. The crudeproduct was purified by flash chromatography (4:1 hexanes/ethyl acetate)and 40 mg of 3-cyclopropyl farnesol was obtained as a colorless oil.

[0081] II. Geranylgeraniol Analogs:

[0082] An illustrative reaction route for synthesizing 3-vinylgeranylgeranyl diphosphate (3-vGPP) and the corresponding 3-vinylgeranylgeraniol is shown in FIG. 5.

[0083] a. Vinyl Analogs

Ethyl 7,11,16-trimethyl-3-oxohexadeca-6(E),10(E),14-trienoate (Compound31, β-keto ester)

[0084] To a suspension of KH (138 mg, 1.2 mmol) in 3.0 ml THF was addeda solution of ethyl acetoacetate (0.064 ml, 0.5 mmol) in 1.0 ml THF at0° C. under argon. After stirring for 20 minutes at room temperature, aclear colorless solution was formed. This solution was cooled to 0° C.and treated with n-butyllithium (1.7 M in hexane, 0.17 ml, 1.1 mmol).After 30 minutes at 0° C., farnesyl bromide (0.285 ml, 1.0 mmol, in 1.0ml of THF) was added to the resulting dianion solution. Stirring wascontinued for an additional 30 minutes. The reaction mixture wasquenched by adding ˜3 ml of 10% aqueous citric acid, and extracted withether (3×15 ml). The organic layers were combined, washed with saturatedNaCl (2×15 ml) and dried over MgSO₄. After purification by flashchromatography (9:1 hexanes/ethyl acetate, R_(f)=0.40), the product 31was obtained as a pale yellow oil (270 mg; 81% yield).

Ethyl3-(trifluoromethylsulfonyloxy)hexadeca-7,11,15-trimethyl-2(Z),6(E),10(E),14-tetraenoate(Compound 32, triflate)

[0085] KH (35% in mineral oil, 178 mg, 1.56 mmol) and 2.0 ml of THF wasplaced in a flame-dried, argon flushed flask. The β-ketoester (260 mg,0.78 mmol) in 1.0 ml of THF was added to this suspension at 0° C. andstirred for 30 minutes. N-(2-Pyridyl)triflimide (349 mg, 0.94 mmol) in1.0 ml of THF was added to the resulting enolate solution at 0° C. Thereaction mixture was stirred at room temperature for 3 hours, quenchedwith ˜5 ml of 10% aqueous citric acid, and extracted with ether (3×15ml). The organic layers were combined, washed with 15 ml of 10% aqueouscitric acid and 15 ml saturated NaCl solution, dried over MgSO₄, andconcentrated. Purification by flash chromatography (20:1 hexanes/ethylacetate) gave 185 mg (58% yield, based on consumed starting material) ofthe triflate (Compound 32 ) as a pale yellow oil, and 30 mg of recoveredβ-ketoester Compound 31.

Ethyl 3-vinyl-7,11,15-trimethylhexadeca-2(Z),6(E),10(E),14-tetraenoate(Compound 33)

[0086] In a flame dried, argon flushed flask were placed triflate (180mg, 0.39 mmol), Pd(PhCN)₂Cl₂ (7.7 mg, 0.02 mmol), AsPh₃ (24 mg, 0.08mmol), CuI (7.4 mg, 0.04 mmol) and 0.5 ml of NMP (99.5%, anhydrous).Vinylbutyltin (0.14 mg, 0.46 mmol) was added to this mixture, andstirred for 15 hrs at room temperature. The reaction mixture was takenup with 100 ml ethyl acetate and washed with aqueous KF (3×30 ml). Theaqueous layer was back-extracted with ethyl acetate (2×15 ml) and thecombined organic layers were dried over MgSO₄. Concentration followed bypurification by flash chromatography (2:1 hexanes/ethyl acetate,R_(f)=0.53) gave Compound 33 as a colorless oil (98 mg, 73% yield). Theidentity, and in particular the stereochemistry, of this ester wasconfirmed by the similarity of its ¹H-NMR spectrum to the previouslyprepared 3-vinyl-3-desmethylfarnesyl ester.

3-Vinyl-7,11,15-trimethylhexadeca-2(Z),6(E),10(E),14-tetraen-1-ol(Compound 34)

[0087] To a solution of ester Compound 33 (95 mg, 0.28 mmol) in 2.0 mlof toluene was added diisobutyl aluminum hydride (1.0 M solution intoluene, 0.7 ml, 0.7 mmol) under argon at −78° C. The reaction wasstirred at −78° C. for one hour and warmed to room temperature. Thereaction was quenched by adding 30 ml of Rochelle salt solution. Theaqueous solution was extracted with ethyl acetate (2×20 ml). Thecombined organic layers were washed with saturated NaCl (2×20 ml) anddried over MgSO₄. Concentration followed by flash chromatography (4:1hexanes/ethyl acetate; R_(f)=0.45) afforded alcohol Compound 34 (52 mg,62% yield) as a colorless oil.

[0088] Compound 35, 3-vGGPP, is made in accordance with the generalprocedures set forth hereinabove for converting the alcohol to thechloride and then to the diphosphate.

[0089] b. Allyl Analogs:

Ethyl 3-allyl-7,11,15-trimethylhexadeca-2(Z),6(E),10(E),14-tetraenoate

[0090] In a flame dried, argon flushed flask were placed triflate 32(562 mg, 1.2 mmol), Pd(PhCN)₂Cl₂ (23 mg, 0.061 mmol), AsPh₃ (38 mg, 0.12mmol), CuI (23 mg, 0.12 mmol) 1.5 ml of NMP (995%, anhydrous). The flaskwas heated to 100° C. To this mixture was added allyltributyltin (0.76ml, 2.4 mmol). After 15 hrs at 100° C., the reaction was cooled, takenup in 100 ml ethyl acetate and washed with aqueous KF (3×30 ml). Theaqueous layer was back-extracted with ethyl acetate (2×15 ml) and thecombined organic layers were dried over MgSO₄. Concentration, followedby purification by flash chromatography (98:2 hexanes/ethyl acetate)gave the ester as a colorless oil (222 mg, 52% yield).

3-Allyl-7,11,15-trimethylhexadeca-2(Z),6(E),10(E),14-tetraen-1-ol

[0091] To the solution of ester (191 mg, 0.53 mmol) in 3.0 ml of toluenewas added diisobutyl aluminum hydride (1.0 M solution in toluene, 1.5ml, 1.5 mmol) under argon at −78° C. The reaction mixture was stirred at−78° C. for one hour and warmed to room temperature. The reaction wasquenched by adding 30 ml of Rochelle salt solution. The aqueous solutionwas extracted with ethyl acetate (2×20 ml). The combined organic layerswere washed with saturated NaCl (2×20 ml) and dried over MgSO₄.Concentration, followed by flash chromatography (9:1 hexanes/ethylacetate) afforded the alcohol 3-allyl-geranylgeraniol (128 mg, 76%yield) as a colorless oil.

3-Allyl-7,11,15-trimethylhexadeca-2(Z),6(E),10(E),14-tetraenediphosphate

[0092] This compound was prepared from alcohol via the correspondingchloride as described above in the general procedures and for 3-vinylGGPP.

[0093] Experimental Procedures and Results

[0094] In Vitro Prenyltransferase Assays:

[0095] FTase IC₅₀ values of seven diphosphate analogs were determinedusing recombinant mFTase (affinity purified rat FPTase) in ascintillation proximity assay with tritiated FPP (specific activity15-30 Ci/mmol, final concentration 0.12 μM) and the peptideBiotin-Aha-Thr-Lys-Cys-Val-Ile-Met-OH (final concentration 0.1 μM) assubstrates. The method is described in detail in Yokama, et al., J.Biol. Chem., Vol. 272, No. 7, pages3944-3952 (Feb. 1997). The FTase,tritiated FPP, and the peptide were incubated at 37° C. for 30 minutesin a buffer. After incubation, a stop reagent and streptavidin beads(Amersham) were added. The radioactive product was counted on a WallacMicrobeta 1450 scintillation counter. The kinetic fits were derived froma nonlinear least squares computer fit of the data. The K_(i) valueswere determined using the same assay system with varying concentrationsof tritiated FPP.

[0096] GGTase I values were determined in a similar manner usingrecombinant mGGTase I, tritiated GGPP, and the peptideBiotin-Aha-Thr-Lys-Cys-Val-Ile-Leu-OH.

[0097] The results of the prenyltransferase assays are shown in Table I:TABLE 1 Inhibition Constants for FPP and GGPP Analogs^(a) Analog IC₅₀FTase IC₅₀ GGTase I K_(i) FTase FTase/GGTase 3-vFPP 173 ˜100,000 96 ˜6003-tbFPP 31 ˜50,000 8.0 ˜1600 3-alFPP 189 >100,000 31 >530 3-etFPP215 >100,000 — >460 3-phFPP 299 >100,000 — >340 3-vGGPP 715 3,050 — 4.33-alGGPP 453 3,380 — 7.5

[0098] Referring to Table 1,3-tbFPP is a potent inhibitor of mFTase. Theselectivity observed for mFTase against the closely related enzymemGGTase I is also noteworthy, and is in accord with the ability ofGGTase I to select for its proper isoprenoid, GGPP. The 3-substitutedFPP analogs inhibited mFTase, albeit not as potently as 3-tbFPP. Thethree most potent inhibitors of mFTase (3-vFPP, 3-tbFPP, and 3-alFPP)were further characterized and were determined to all be competitiveinhibitors of the enzyme versus FPP.

[0099] The geranylgeranylase analogs 3-vGGP and 3-alGGPP were surprisingin their ability to bind more tightly to mFTase than to mGGTase I. Thisunderscores the surprising difference in diphosphate binding selectivitybetween these two enzymes which are highly similar in amino acidsequence, and in fact, share an identical a subunit. It also emphasizesthe highly selective nature of GGTase I and the difficulty in obtainingeither peptide- or isoprenoid-based inhibitors of this enzyme that donot also inhibit mFTase.

[0100] Soft Agar Assays to Demonstrate Inhibition of Ras-transformedCell Growth In Vitro:

[0101] The isoprenyl diphosphate analogs are unstable and unlikely topenetrate cell membranes unaided, although the natural isoprenoids areapparently taken up by cells through an active transport system. Sinceit has been demonstrated that mammalian cells can utilize farnesol andgeranylgeraniol for the prenylation of proteins, the 3-substitutedisoprenoid alcohol analogs were used in this study to inhibit theanchorage-independent growth of transformed NIH3T3 fibroblasts.Presumably, the non-polar alcohols pass through the cell membrane andare then diphosphorylated by an as yet uncharacterized kinase to FPP orGGPP.

[0102] The 3-substituted analogs were evaluated as inhibitors of thegrowth of ras-transformed cells by comparing their relative degree ofactivity against an isogenic panel of transformed cell lines, comprisedof NIH 3T3 fibroblasts transfected with either H-Ras(61L) [H-Ras-F],H-Ras(61L)CVLL [H-Ras-GG], or a transforming form of c-raf [Raf]. TheH-Ras-F, H-Ras-GG, and raf transformed NIH3T3 cell lines have beenpreviously described in Cox, et al., Molecular and Cellular Biology,Vol. 12, No. 6, pages 2606-2615 (1992). The cells were grown prior toplating in Dulbecco's modified Eagle medium supplemented with 10% calfserum and 1% antibiotic/antimycotic at 37° C. and 10% CO₂. Experimentswere carried out in 6-well dishes in a two layer agar system (0.6%bottom layer and 0.3% top layer). Cells were incorporated into the toplayer along with varying concentrations of the compound prepared inethanol. Compound addition occurred only at the time the cells wereseeded. Subsequent incubation was at 37 ° C. with 10% CO₂ for 2 weeks.Colonies were stained with 0.5 ml/well of 1 mg/ml p-iodonitrotetrazoliumviolet (Sigma) for 24 hours prior to quantitation by image analysis.

[0103] The results are shown below in Table 2. Of the six analogstested, only the vinyl and allyl compounds exhibited cellular activity.Although not shown on Table 2, 3-tert-butylfarnesol proved to beinactive in cells, which may be due to the inability of the putativekinase to accept the bulky tert-butyl-substituted alcohol. TABLE 2Anchorage-Independent Growth Inhibition by Farnesol and GeranylgeraniolAnalogs^(a) Mean IC₅₀ (± S.E.) (μM) Analog H-Ras-F H-Ras-GG Raf 3-vinylFarnesol 10.9 ± 2.6 (8) 14.1 ± 1.4 (5)    1.9 (1.3, 2.5) 3-ally Farnesol10.2 ± 3.5 (3) >25 (4)   13.2 (10.3, 16.0) 3-ethyl Farnesol >25 (2) >25(2) n.d. 3-phenyl Farnesol >25 (2) >25 (2) n.d. 3-vinyl 18.0 ± 4.1 (5)13.9 ± 2.3 (4)    5.6 (4.7, 6.4) Geranylgeraniol 3-allyl >25 (4)  4.6 ±1.9 (3)   >25 (2) Geranylgeraniol

[0104] The most striking selectivity was observed for3-allylgeranylgeraniol, which exhibited an IC₅₀ of 4.6 μM againstH-Ras-GG cells, but was totally ineffective (IC₅₀>25 μM) against bothH-Ras-F and Raf cells. In contrast, 3-allylfarnesol exhibited the samedegree of activity against the H-Ras-F and Raf lines, although it didprove to be ineffective against H-Ras-GG cells. When comparing thebiological activities of the various analogs, H-Ras-F and Raf cells wereuniformly more susceptible to 3-vinylfarnesol than3-vinylgeranylgeraniol, whereas H-Ras-GG cells were equally inhibited byboth compounds.

[0105] Subcellular Fractionation Experiments:

[0106] The soft agar data presented hereinabove established the 3-vinyland 3-allyl alcohol analogs as potent inhibitors of transformed cellgrowth. Surprisingly, neither 3-vinylfarnesol nor 3-vFPP blocked theprenylation of Ras proteins in H-Ras-F cells. This is contrary to theresults seen with typical FTase inhibitors. Since the mechanism for theinhibition effect has not been firmly established, the ability of thecorresponding diphosphates (3-vFPP and 3-alFPP) to inhibit FTase in vivowas investigated in a series of subcellular fractionation experiments.

[0107] H-Ras-F (A) or H-Ras-GG (B) cells were treated with 25 μMlovastatin for 24 h after which the indicated FPP or GGPP analogs(resuspended in media) were added directly to the cell media. Followingan additional 24 h incubation period, the cells were harvested, lysedand the membranes (M) separated from the cytosol (C). Aftersolubilization of the membrane fraction, Ras protein wasimmunoprecipitated from both the membrane and cytosolic fractions by theaddition of the Y13-259 antibody (OP04 from Oncogene Science). Thepresence of Ras protein in each fraction was analyzed by Westerntransfer techniques. Incubation with the primary antibody (pan-ras Ab-2from Oncogene Science) and an anti-mouse HRP conjugate secondaryantibody (Amersham) was employed for detection of the ras protein. Blotswere developed using Enhanced Chemiluminescence techniques (Amersham).The technique is described in detail by Scholten, et al., Bioorg. Med.Chem., Vol. 4, pages 1537-1543 (1992).

[0108] The results are shown in FIGS. 6 and 7 which are Western blots ofthe H-Ras-F and H-Ras-GG experiments, respectively. H-Ras-F cells weretreated with lovastatin in order to block the mevalonate pathway,prevent the formation of FPP and GGPP, and thus, inhibit proteinprenylation. This inhibition is evidenced on the Western blot shown inFIG. 6 by a shift of the majority of H-Ras from the membrane fraction(M) to the cytosol (C), when compared with the DMSO control. The shiftis significantly reversed by subsequent treatment of the cells with FPP.Dosing of the lovastatin-treated cells with 3-alFPP results in virtuallyall of the H-Ras being found in the cytosol. This is consistent with3-alFPP acting as an FTase inhibitor rather than a substrate. In sharpcontrast, dosing of the lovastatin-treated cells with 3-vFPP results invirtually complete localization of the H-Ras protein in the membranefraction (M). Thus, it appears that 3-vFPP acts as an alternativesubstrate for FTase, leading to the formation of 3-vinylfarnesylatedRas.

[0109] Further confirmation of these results was provided by treatmentof H-Ras-F cells with tritium-labeled 3-vinylfarnesol. H-Ras-F cellswere treated with 25 μM lovastatin for 24 h after which the followingwere added directly to four samples of the cell media: (1)control—untreated, (2) 3-vinyl-1-[³H]-farnesol (5 μM, 1.34 μCi/ml), (3)1-[³H]-FPP (3 μM, 50 μCi/ml; Amersham), and (4) 1-[³H]-farnesol (3 μM,50 μCi/ml; American Radiolabeled Chemicals). Following an 18 hincubation period, the cells were lysed and the proteins were separatedby SDS-PAGE on a 14% gel, and transferred to an Immobilon-P PVDFmembrane (Millipore). After drying, the membranes were sprayed withEn³Hance (Amersham) and exposed to film (Hyperfilm MP, Amersham) for aperiod of time prior to developing (4 days for 1-[³H]-FPP and1-[³H]-farnesol) and 28 days for 3-vinyl-1-[³H]-farnesol). The exposedfilms are shown on FIG. 8 where the control and radioactive bands aredesignated as indicated above. Referring to FIG. 8, the radiolabelmigrates with the same protein bands as seen when cells are treated withtritiated farnesol and FPP, verifying the farnesylation of theseproteins by 3-vFPP.

[0110] The selectivity and mechanism of the observed cell growthinhibition was further probed by studying the effects of the 3-vinyl and3-allyl GGPP analogs on the subcellular distribution of thegeranylgeranylated protein variant in H-Ras-GG cells in a manner similarto that described for 3-vFPP and 3-alFPP. The Western blot is shown onFIG. 8. With H-Ras-GG cells, as with H-Ras-F cells, blockage of themevalonate pathway results in a shift in the subcellular location of theRas protein from the membrane to the cytosol. In accord with the resultsdescribed above, dosing of lovastatin-treated H-Ras-GG cells with3-vGGPP, but not 3-alGGPP, resulted in restoration of the membranelocalization of H-Ras-GG.

[0111] It has been well established that levels of lovastatin thatcompletely block the mevalonate pathway result in cell cycle blockade,significant cytotoxicity, and a sharp change in cell appearance to arounded morphology. The effects of lovastatin on the cell morphology ofH-Ras-F and H-Ras-GG cells treated with FPP, GGPP, 3-vFPP, 3-vGGPP,3-alFPP, and 3-al GGPP were photographed and the results are shown onFIG. 9 (H-Ras-F) and 8 (H-Ras-GG), respectively. The H-Ras-F andH-Ras-GG cells were grown in six-well plates to 70-80% confluence. Aftertreating the cells with lovastatin (25 μM) for 24 hour, the indicatedFPP or GGPP analogs (re-suspended in media) were added directly to thecell media. Photographs were taken after an additional 24 hourincubation period.

[0112] The change in cell morphology can be reversed inlovastatin-treated H-Ras-F cells with geranylgeraniol or GGPP, but notfarnesol or FPP, as demonstrated in FIG. 9. As expected, 3-alFPP was notable to reverse the morphology of lovastatin-treated cells; however,3-vFPP exhibits a modest ability to do so. The ability of 3-vGGPP torestore the flattened morphology of cells is in accord with its abilityto prenylate H-Ras-GG (See FIG. 7). However, the reversion in morphologyseen with 3-alGGPP was completely unexpected. Furthermore, withlovastatin-treated H-Ras-GG cells, 3-vGGPP was able to completelyrestore, and 3-alGGPP was able to partially restore, the flattenedmorphology (FIG. 10). This indicates that 3-alGGPP can be used as aprenyl source by GGTase I with certain protein substrates, but notH-Ras(61L)CVLL. An alternative explanation is that 3-alGGPP may serve asa substrate for GGTase II, and prenylation of these proteins may allowfor morphological reversion of cells.

[0113] In conclusion, the data presented herein demonstrate that certainFPP analogs can act as potent inhibitors of mammalian FTase; farnesoland geranylgeraniol analogs can be prodrugs for the corresponding FPPand GGPP derivatives; and the prenyl alcohol derivatives potentlyinhibit the growth of ras-transformed cells via two differentmechanisms. The selectivity of 3-allylfarnesol and3-allylgeranylgeraniol in soft agar assays (Table 2) and their behaviorin the subcellular fractionation experiments (FIGS. 6 and 7) are inaccord with previous studies on FTase and GGTase I inhibitors. That is,they appear to block the growth of ras-transformed cells by preventingthe prenylation of the Ras protein. It is striking and surprising,however, that 3-allylgeranylgeraniol exhibits such selectivity in cells,while 3-alGGPP exhibits no selectivity in vitro (see Table 1). Perhapsthe intracellular level of GGPP is in the low nanomolar level incontrast to the much higher intracellular FPP concentration (˜5 μM), andthus 3-alGGPP can compete effectively with the natural substrate forGGTase I but not FTase. Nevertheless, 3-allylgeranylgeraniol is a highlyspecific cellular inhibitor of protein geranylgeranylation, and thus mayalso be a valuable tool to investigate the relative biologicalimportance of geranylgeranylation versus farnesylation.

[0114] In sharp contrast, 3-vinylfarnesol is converted to 3-vFPP, whichacts as an alternative substrate for FTase and serves as a prenyl donorboth in vitro and in vivo. The corresponding geranylgeranyl analogappears to act in the same manner. Thus, the observed biologicalactivity of the vinyl substituted compounds is not due to FTase orGGTase I inhibition. While not wishing to be bound by theory, theactivity could be due to inhibition of squalene synthase,cis-prenyltransferase, or trans-prenyltransferase, which utilize FPP tomake cholesterol, dolichol and ubiquinone, respectively. Relatively highconcentrations of farnesol have antiproliferative effects on culturedtumor cells. However, in control experiments 30 μM farnesol exhibitedlittle effect on the proliferation of H-Ras-F cells (data not shown), incontrast to the complete inhibition of growth seen with 3-vinylfarnesol.In the studies presented below, 500 nM farnesol had no effect on HPACcells whereas 500 nM 3-vinylfarnesol and 3-allylfarnesol were potentlycytotoxic against HPAC cells in situ.

[0115] Effect of 3-Substituted Farnesols on the Proliferation of HPACCells.

[0116] The malignant human cell line HPAC (human pancreaticadenocarcinoma cells) was used in this study. The cells were seeded at aconcentration of 2×10⁵/ml in 24 well culture plate (Costar, Cambridge,Mass.). 3-Vinylfarnesol and 3-allylfarnesol were dissolved separately in95% ethanol to make a stock solution. Varying amounts of the stocksolutions were then used to treat the HPAC cells. The finalconcentration of the farnesol analogs in the culture plate wells rangedfrom 0 nM (ethanol-treated control) to 500 nM. The plate was thenincubated at 37° C. under a 5% CO₂-humidified atmosphere for 48 hours.The total viable cells from each well were determined by Trypan Blue(0.4%) exclusion (Gibco, N.Y.) followed by cell counting. Under theseconditions, 2.×10⁶ cells were found in the control well. With both3-substituted analogs, treatment with 100 nM of the analog led tocytostasis (the cell count was roughly equal to 2×10⁵/well) whiletreatment with 500 nM of the analog led to complete cytotoxicity (noviable cells remained in the well). Under the same conditions, 500 nM ofthe natural isoprenoid farnesol had no effect on the growth of HPACcells, leading to the same cell count as the ethanol-treated control.

[0117] Preliminary In Vivo Toxicity Studies

[0118] 3-Vinylfarnesol and 3-allylfarnesol were dissolved in 95% ethanoland then diluted in normal saline. A 0.1 mg/kg dose of 3-vinylfarnesolwas injected into a SCID mouse. No toxic response was seen after 48hours, so a further 1 mg/kg dose was injected into the same mouse.Again, no adverse effects were seen after 48 hours, so an additional 10mg/kg dose was given. No visible adverse effects were seen at any pointduring this treatment.

[0119] A 0.1 mg/kg dose of 3-allylfarnesol was injected into a secondSCID mouse. No toxic response was seen after 48 hours, so a further 1mg/kg dose was injected into this mouse. Again, no adverse effects wereseen after 48 hours, so an additional 10 mg/kg dose was given. Novisible adverse effects were seen at any point during this treatment.

[0120] Although the invention has been described in terms of specificembodiments and applications, persons skilled in the art can, in lightof this teaching, generate additional embodiments without exceeding thescope or departing from the spirit of the claimed invention.Accordingly, it is to be understood that the drawing and description inthis disclosure are proffered to facilitate comprehension of theinvention, and should not be construed to limit the scope thereof.

What is claimed is:
 1. A compound of the general formula:

wherein R′ is a substituted or unsubstituted C₁₀-C₂₀ saturated orunsaturated alkyl, aryl, heteroaryl or cycloalkyl; R is a substituted orunsubstituted C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group; and X is —OH or —P₂O₇. 2.The compound of claim 1 wherein R′ is geranyl.
 3. The compound of claim1 wherein R′ is farnesyl.
 4. The compound of claim 1 wherein R′ isgeranyl or farnesyl and R is selected from the group consisting ofvinyl, ethyl, allyl, saturated and unsaturated isomers of propyl, butyl,and pentyl, cyclopropyl, cyclopentyl, phenyl and heterosubstitutedmoieties, such as fluorophenyl, (trimethylsilyl)methyl, 1-ethoxyvinyl,and 2-furanyl, 2-thiophenyl.
 5. The compound of claim 1 which is7-allylfarnesol.
 6. The compound of claim 1 which ispara-biphenylfarnesol.
 7. A compound of the general formula:

wherein R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, orcycloalkyl group, or a C₆-C₁₀ aromatic groups or heteroaromatic group,and X is —OH or —P₂O₇.
 8. The compound of claim 7 wherein R is selectedfrom the group consisting allyl, saturated and unsaturated isomers ofpropyl, butyl, and pentyl, cyclopentyl, phenyl and heterosubstitutedmoieties, such as fluorophenyl, (trimethylsilyl)methyl, 1-ethoxyvinyl,and 2-furanyl, 2-thiophenyl.
 9. The compound of claim 7 which is3-allylfarnesol.
 10. The compound of claim 7 which is 3-allyl farnesyldiphosphate.
 11. The compound of claim 7 which is 3-isopropylfarnesol.12. The compound of claim 7 which is 3-isopropylfarnesyl diphosphate.13. A compound of the general formula:

wherein R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, orcycloalkyl group, or a C₆-C₁₀ aromatic groups or heteroaromatic group,and X is —OH or —P₂O₇.
 14. The compound of claim 13 wherein R isselected from the group consisting allyl, saturated and unsaturatedisomers of propyl, butyl, and pentyl, cyclopentyl, phenyl andheterosubstituted moieties, such as fluorophenyl,(trimethylsilyl)methyl, 1-ethoxyvinyl, and 2-furanyl, 2-thiophenyl. 15.The compound of claim 13 which is 3-allylgeranylgeraniol.
 16. Thecompound of claim 13 which is 3-allyl farnesyl diphosphate.
 17. Atherapeutic composition comprising: a 3-substituted isoprenol analog ofthe general formula:

wherein R′ is a substituted or unsubstituted C₁₀-C₂₀ saturated orunsaturated alkyl, aryl, heteroaryl or cycloalkyl; R is a substituted orunsubstituted C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group; and X is —OH or —P₂O₇; and apharmaceutically acceptable carrier.
 18. The therapeutic composition ofclaim 17 wherein R is selected from the group consisting of vinyl,ethyl, allyl, saturated and unsaturated isomers of propyl, butyl, andpentyl, cyclopropyl, cyclopentyl, phenyl and heterosubstituted moieties,such as fluorophenyl, (trimethylsilyl)methyl, 1-ethoxyvinyl, and2-furanyl, 2-thiophenyl.
 19. A method of treating cancer comprisingadministering an effective amount of at least one 3-substitutedisoprenol derivative to a patient having a cancer of the type that issusceptible to treatment with a 3-substituted isoprenol derivativehaving the general formula:

wherein R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group.
 20. The method of claim 19wherein the 3-substituted isoprenol derivative is selected from thegroup consisting of 3-vinyl farnesol, 3-allylfarnesol,3-isopropylfarnesol, 3-vinyl geranylgeraniol, and3-allylgeranylgeraniol.
 21. The method of claim 19 wherein the cancer ishuman pancreatic cancer.
 22. The method of claim 19 wherein the canceris human colon cancer.
 23. A method for reducing the level of proteinfarnesylation in mammalian cells in a mammalian host, wherein said cellsare sensitive to treatment with a compound with the formula:

wherein R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group, and X is —OH or —P₂O₇; thesaid method comprising administering to said mammalian host an amount ofthe compound effective to inhibit the activity of farnesyl proteintransferase; wherein the activity of farnesyl protein transferase isreduced.
 24. The method of claim 23 wherein the compound is selectedfrom the group consisting of 3-vinyl farnesol, 3-allylfarnesol,3-isopropylfarnesol, 3-vinyl geranylgeraniol, and3-allylgeranylgeraniol.
 25. A method for reducing the level of proteingeranylgeranylation in mammalian cells in a mammalian host, wherein saidcells are sensitive to treatment with a compound with the formula:

wherein R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group, and X is —OH or —P₂O₇; thesaid method comprising administering to said mammalian host an amount ofthe compound effective to inhibit the activity of geranylgeranyl proteintransferase; wherein the activity of geranylgeranyl protein transferaseis reduced.
 26. The method of claim 25 wherein the compound is selectedfrom the group consisting of 3-vinyl geranylgeraniol and3-allylgeranylgeraniol.
 27. A method of reducing the proliferation oftumor cells of the type that are sensitive to treatment with a3-substituted isoprenol analogs of the formula:

wherein R is a C₂-C₁₀ saturated or unsaturated alkyl, aryl, cycloalkyl,or C₆-C₁₀ aromatic or heteroaromatic group, and X is —OH or —P₂O₇, themethod comprising subjecting the tumor cells to an amount of the3-substituted isoprenol derivatives sufficient to act as a competitiveinhibitor of prenyl protein transferase thereby reducing theproliferation of the tumor cells.
 28. The method of claim 27 wherein themammalian cells are tumor cells that are associated with abnormalactivity of oncogenes in the ras family or its pathway.
 29. The methodof using of 3-allyl farnesyl or 3-vinyl farnesyl diphosphate-basedfarnesyl transferase inhibitors as probes for analyzing the FPP-bindingsite of FTase.
 30. The method of using of 3-allyl geranylgeranyl or3-vinyl geranylgeranyl diphosphate-based geranylgeranyl transferaseinhibitors as probes for analyzing the GPP-binding site of GGTase.
 31. Amethod of preventing restenosis by administering an effective amount of3-allyl or 3-vinyl geranylgeraniol to a patient in need of treatment forrestenosis following cardiac catheterization or angioplasty.